The family of polo-like kinase (PLK) proteins are central players in regulating entry into and progression through mitosis. The four known human PLKs have non-redundant and non-overlapping functions. A significant body of literature has validated PLKs as anti-tumor drug targets and suggested that profound anti-proliferative activity is achieved through selective inhibition of PLK1 functions. Over-expression of PLK1 is frequently observed and PLK1 expression is a prognostic indicator for outcome of patients suffering from various tumors. For example, more than half of prostate cancers over-express PLK1 and this expression is positively correlated with tumor grade. PLK1 is also extensively over-expressed in colorectal cancer and recently has been demonstrated to be a potential therapeutic target in colorectal cancer cell lines with inactivated p53. Moreover, it has been reported that p53 transcriptionally regulates PLK1 expression, providing more direct evidence that PLK1 is oncogenic when p53 is mutated. Thus, there is a strong rationale for pursuing PLK1 as an anti-tumor drug target. Indeed, the therapeutic rationale for PLK inhibition has been validated through studies with PLK1-specific antisense oligonucleotides and shown to profoundly induce growth inhibition in cancer cells both in vitro and in vivo.
Numerous inhibitors of ATP binding to the catalytic site in the N-terminal region of the PLKs have been identified with some entering clinical trials after showing significant anti-tumor activity in preclinical models. At least two compounds have been evaluated in phase I clinical trials. Published results from these two compounds suggest acceptable toxicity profiles thus warranting further investigation in phase II trials.
The human PLK family has four members that play a key role in mitotic progression and DNA integrity. Of the four family members, PLK1 is the best-characterized. A significant potential drawback of compounds that target the PLK1 ATP cleft is that other members of the PLK family are likewise targeted by the compounds. For instance, at least 3 of the 4 known members of the mammalian PLKs are inhibited by BI2536, the most advanced PLK inhibitor found to date. Contrary to the role of PLK1 in cell proliferation and tumorigenesis, its two most closely related family members, PLK2 and PLK3, appear to play a role in check-point mediated cell-cycle arrest. Since PLK3 has been reported to have opposing functions to PLK1 (collective evidence indicates that PLK3 acts as a tumor suppressor), inhibition of this kinase may lead to diminution of the anti-tumor effect mediated by blocking PLK1 activity. For instance, it has been shown that mice deficient in PLK3 were susceptible to tumors in various organs and that tumor suppressor activity could be directly attributed to the catalytic domain. It has also been demonstrated that PLK3 positively regulates the PTEN tumor suppressor. These issues were revealed subsequent to the initial clinical development of ATP-competitive PLK inhibitors and strongly suggest that inhibiting PLK3 would not be a desirable feature of a clinical candidate. In addition, ATP competitive inhibitors will only block the enzymatic functions of PLK1 and will not necessarily have an effect against other critical functions in mitosis.
The two domains found in the human PLK1 are the highly-conserved N-terminal kinase domain and the unique polo box domain (PBD) containing two polo boxes. The PBD involves phosphopeptide binding, which helps regulate substrate binding at the kinase catalytic domain. Previous studies with peptides provide evidence that the substrate and sub-cellular targeting binding site in the PBD forms a compact and druggable interface amenable to inhibitor development. Although high-throughput screening approaches have identified small molecule inhibitors of the PBD-peptide interaction, these either are weakly binding or non-drug-like in nature. In addition, these inhibitors display a contrasting phenotype to PLK1 knockdown and to cellular treatment with inhibitors of catalytic activity. Peptide inhibitors have also been found that can target the PBD domain and discriminate between the PLK family members. Unfortunately, however, while peptides make excellent inhibitors, they generally exhibit poor pharmacokinetic properties and as such have not proven useful in clinical settings.
What are needed in the art are alternative approaches to develop potent and highly selective PLK1 inhibitors and PLK1 inhibitors developed by such methods. A PLK1 selective inhibitor could beneficially avoid inhibition of the tumor suppressor activity of PLK2 and PLK3. In addition, a PLK1 inhibitor that targets the distinctive binding site on the PBD could be used to generate novel anticancer therapeutics that are both discriminatory to PLK1 and less toxic to normal cells.